The simultaneous acquisition of resistance to multiple structurally and chemically unrelated compounds often involves active membrane efflux pumps, referred to as multidrug (MDR) transporters. Multidrug transporters have been implicated in drug resistance in bacteria, in the modulation of drug clearance in the human kidney and liver, and in compromising the effectiveness of chemotherapy in cancer treatment. The long term goal of this proposal is to define the conformational motion that transduces energy input into the mechanical work of substrate translocation in MDR transporter superfamilies. Our approach emphasizes state-of-the-art electron paramagnetic resonance (EPR) methods in conjunction with mutagenic analysis to define the nature and amplitude of ligand-dependent structural rearrangements, reveal conformational equilibria, identify substrate binding sites and permeation pathways, and decode the principles of ion and substrate coupling, all in the native-like environment of the lipi bilayer. Over the last two funding cycles, we described the dynamics of ATP-powered alternating access of ABC transporters and uncovered a proton-activated structural switch in secondary MDR transporters from the major facilitator superfamily (MFS). One of the goals in the next funding period is to elucidate the structural mechanisms of alternating access for the recently characterized class of multidrug and toxic compound extrusion (MATE) transporters which have been associated with bacterial resistance to a new generation of antibiotics and implicated in drug disposition for humans. Aim 1 and 2 will carry out comparative analysis of two classes of Na+- and H+- coupled MATE transporters to delineate the basis of mechanistic diversity in this superfamily. Another goal is to investigate how lipids shape the conformational cycle of MDR transporters of the major facilitator superfamily. Grounded in progress in the previous funding period, aim 3 will test the hypothesis that specific interactions between conserved sequence motifs and lipids modulate the isomerization of MFS-MDR transporters during transport.